This invention relates generally to nucleic acid sequences encoding proteins that are associated with abiotic stress responses and abiotic stress tolerance in plants. In particular, this invention relates to nucleic acid sequences encoding proteins that confer drought, heat, cold, and/or salt tolerance to plants.
Abiotic environmental stresses, such as drought stress, salinity stress, heat stress, and cold stress, are major limiting factors of plant growth and productivity (Boyer. 1982. Science 218, 443-448). Crop losses and crop yield losses of major crops such as rice, maize (corn) and wheat caused by these stresses represent a significant economic and political factor and contribute to food shortages in many underdeveloped countries.
Plants are typically exposed during their life cycle to conditions of reduced environmental water content. Most plants have evolved strategies to protect themselves against these conditions of low water or desiccation (drought). However, if the severity and duration of the drought conditions are too great, the effects on plant development, growth and yield of most crop plants are profound. Continuous exposure to drought causes major alterations in the plant metabolism. These great changes in metabolism ultimately lead to cell death and consequently yield losses.
Developing stress-tolerant plants is a strategy that has the potential to solve or mediate at least some of these problems (McKersie and Leshem, 1994. Stress and Stress Coping in Cultivated Plants, Kluwer Academic Publishers). However, traditional plant breeding strategies to develop new lines of plants that exhibit resistance (tolerance) to these types of stresses are relatively slow and require specific resistant lines for crossing with the desired line. Limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding. Additionally, the cellular processes leading to drought, cold and salt tolerance are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways (McKersie and Leshem, 1994. Stress and Stress Coping in Cultivated Plants, Kluwer Academic Publishers). This multi-component nature of stress tolerance has not only made breeding for tolerance largely unsuccessful, but has also limited the ability to genetically engineer stress tolerance plants using biotechnological methods.
Drought, heat, cold and salt stresses have a common theme important for plant growth and that is water availability. Plants are exposed during their entire life cycle to conditions of reduced environmental water content. Most plants have evolved strategies to protect themselves against these conditions. However, if the severity and duration of the drought conditions are too great, the effects on plant development, growth and yield of most crop plants are profound. Since high salt content in some soils result in less available water for cell intake, its effect is similar to those observed under drought conditions. Additionally, under freezing temperatures, plant cells loose water as a result of ice formation that starts in the apoplast and withdraws water from the symplast (McKersie and Leshem, 1994. Stress and Stress Coping in Cultivated Plants, Kluwer Academic Publishers). Commonly, a plant's molecular response mechanisms to each of these stress conditions are common.
The results of current research indicate that drought tolerance is a complex quantitative trait and that no real diagnostic marker is available yet. High salt concentrations or dehydration may cause damage at the cellular level during drought stress but the precise injury is not entirely clear (Bray, 1997. Trends Plant Sci. 2, 48-54). This lack of a mechanistic understanding makes it difficult to design a transgenic approach to improve drought tolerance. However, an important consequence of damage may be the production of reactive oxygen radicals that cause cellular injury, such as lipid peroxidation or protein and nucleic acid modification. Details of oxygen free radical chemistry and their reaction with cellular components such as cell membranes have been described (McKersie and Leshem, 1994. Stress and Stress Coping in Cultivated Plants, Kluwer Academic Publishers).
There are numerous sites of oxygen activation in the plant cell, which are highly controlled and tightly coupled to prevent release of intermediate products (McKersie and Leshem, 1994. Stress and Stress Coping in Cultivated Plants, Kluwer Academic Publishers). Under abiotic stress situations, it is likely that this control or coupling breaks down and the process “dysfunctions” leaking activated oxygen. These uncoupling events are not detrimental provided that they are short in duration and that the oxygen scavenging systems are able to detoxify the various forms of activated oxygen. If the production of activated oxygen exceeds the plant's capacity to detoxify it, deleterious degenerative reactions occur. At the subcellular level, disintegration of membranes and aggregation of proteins are typical symptoms. Therefore it is the balance between the production and the scavenging of activated oxygen that is critical to the maintenance of active growth and metabolism of the plant and overall environmental (abiotic) stress tolerance.
Preventing or diminishing the accumulation of oxygen free radicals in response to drought is a potential way to engineer tolerance (Allen, 1995. Plant Physiol. 107, 1049-1054). Overexpression of antioxidant enzymes or ROS-scavenging enzymes is one possibility for the induction of functional detoxification systems. For example, transgenic alfalfa plants expressing Mn-superoxide dismutase tend to have reduced injury after water-deficit stress (McKersie et al., 1996. Plant Physiol. 111, 1177-1181). These same transgenic plants have increased biomass production in field trials (McKersie et al., 1999. Plant Physiology, 119: 839-847; McKersie et al., 1996. Plant Physiol. 111, 1177-1181). Transgenic plants that overproduce osmolytes such as mannitol, fructans, proline or glycine-betaine also show increased resistance to some forms of abiotic stress and it is proposed that the synthesized osmolytes act as ROS scavengers (Tarczynski. et al. 1993. Science 259, 508-510; Sheveleva, et al. 1997. Plant Physiol. 115, 1211-1219). Overexpression of glutathione reductase has increased antioxidant capacity and reduced photoinhibition in popular trees (Foyer et al., 1995. Plant Physiology 109: 1047-57).
The glutaredoxin and thioredoxin proteins are small heat-stable oxidoreductases that have been conserved throughout evolution. They function in many cellular processes, including deoxyribonucleotide synthesis, protein folding, sulfur metabolism and most notably repair of oxidatively damaged proteins. They have also been implicated in the regulation of redox homeostasis in the cell and redox potential has been implicated in changes in gene expression.
Thioredoxins have a dithiol/disulfide (CGPC) at their active site and are the major cellular protein disulfide reductases. Cytosolic isoforms are present in most organisms. Mitochondria have a separate thioredoxin system and plants have chloroplast thioredoxins, which regulate photosynthetic enzymes by light via ferredoxin-thioredoxin reductase. Thioredoxins are critical for redox regulation of protein function and signaling via thiol redox control. Several transcription factors require thioredoxin reduction for DNA binding (Arner and Holmgren, 2000. European Journal of Biochemistry 267: 6102-6109; Spyrou et al., 2001. Human Genetics 109: 429-439).
Glutaredoxins are small heat-stable proteins that are active as glutathione-dependent oxidoreductases. They catalyze glutathione-disulfide oxidoreductions overlapping the functions of thioredoxins and using reducing equivalents from NADPH via glutathione reductase. In Saccharomyces cerevisiae, two genes, GRX1 and GRX2, whose expression is induced in response to various stress conditions including oxidative, osmotic, and heat stress, encode glutaredoxins. Furthermore, both genes are activated by the high-osmolarity glycerol pathway and negatively regulated by the Ras-protein kinase (Grant C M. 2001. Molecular Microbiology 39: 533-541; Grant C M et al., 2001. Biochimica et Biophysica Acta—Gene Structure & Expression 1490: 33-42).
Another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) differs from the first in containing a single cysteine residue at the putative active site (Rodriguez-Manzaneque et al., 1999. Molecular & Cellular Biology 19: 8180-8190). The role of these enzymes is not fully understood.
In addition to the two gene pairs encoding cytoplasmic glutaredoxins (GRX1, GRX2), Saccharomyces cerevisiae also contains two gene pairs for thioredoxins (TRX1, TRX2). Only a quadruple mutant is non-viable and either a single glutaredoxin or a single thioredoxin can sustain viability, indicating some cross function between the two systems (Draculic et al., 2000. Molecular Microbiology 36: 1167-1174).
Plants also contain glutaredoxins genes. A glutaredoxin (thioltransferase), which catalyzes thiol/disulfide exchange reaction, was isolated from rice (Oryza saliva L.) (Sha et al., 1997. Journal of Biochemistry 121: 842-848; Sha et al., 1997. Gene 188: 23-28; GenBank accession number D86744). Mulitple forms of glutaredoxin have also been predicted in the Arabiposis genome (GenBank).
Dehydroascorbate reductase (DHAR; glutathione: dehydroascorbate oxidoreductase, EC 1.8.5.1) is an enzyme that is critical for maintenance of an appropriate level of ascorbate in plant cells by the cycling of dehydroascorbate to replenish ascorbate. DHAR was considered a specific enzyme of the ascorbate-glutathione cycle. However, at least four distinct proteins can catalyze in vitro both glutathione-dependent DHA reduction and other reactions mainly related to thiol-disulphide exchange. These glutaredoxin enzymes (thioltransferases) have both thiol-disulfide oxidoreductase and dehydroascorbate reductase activities (Kato et al., 1997. Plant & Cell Physiology 38: 173-178; Detullio et al., 1998. Plant Physiology & Biochemistry 36: 433-440). Therefore glutaredoxins may also function in vivo as DHAR.
There have been no reports on the mutation or overexpression of either thioredoxin or glutaredoxin in plant cells to determine their function in terms of oxidative stress tolerance or drought tolerance.